Soy phytoestrogens: impact on postmenopausal bone loss and mechanisms of action

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1 Lead Article Soy phytoestrogens: impact on postmenopausal bone loss and mechanisms of action Raewyn C Poulsen and Marlena C Kruger Due to their ability to mimic the actions of mammalian estrogens, soy phytoestrogens have been proposed as potential therapeutic agents to aid in preventing postmenopausal bone loss. In vitro, phytoestrogens promote osteoblastogenesis and inhibit osteoclastogenesis. Although a relatively large number of intervention studies have been undertaken in animals and humans, the efficacy of phytoestrogens as bone-protective agents in vivo remains unclear. Differences in the bioactivities of individual phytoestrogens, differences in phytoestrogen metabolism and bioavailability within different study populations, and imprecise reporting of the dose of phytoestrogens administered in intervention studies may have contributed to the disparity in study findings International Life Sciences Institute INTRODUCTION Phytoestrogens are plant-derived compounds that exhibit similar, albeit weaker, effects than mammalian estrogens. Menopause in women results in reduced production of endogenous estrogens. Estrogens play a major role in the regulation of bone metabolism and some of the consequences of menopause are reductions in bone mass and increased risk of osteoporosis. There is interest in determining the efficacy of phytoestrogens as a possible means of minimizing postmenopausal bone loss in women. In Western countries, approximately 30% of women over the age of 50 years have osteoporosis. 1 Osteoporosis is a debilitating disease characterized by decreased bone mass, microarchitectural deterioration of bone tissue, and increased risk of fracture. 2 It arises due to disruption of the normal process of bone remodeling. Bone remodeling occurs throughout the lifetime. It involves the sequential and coupled resorption of fatigued bone tissue and replacement with newly synthesized bone. Bone remodeling ensures the structural integrity of the skeleton is maintained. Three different types of bone cell osteocytes, osteoclasts, and osteoblasts are involved in the remodeling process. Osteocytes are cells that reside within bone. They sense mechanical stress and signal the need for remodeling at specific sites in bone. 3 In response to osteocyte signaling, osteoclasts resorb fatigued bone tissue. 4 New bone is then synthesized and laid down by osteoblasts. 5 Usually, osteoclast-mediated bone resorption is balanced completely by osteoblastmediated bone formation. However, lifestyle factors, endocrine changes, such as those that occur with aging, or inflammation, which may arise as a result of illness, impact the regulation of bone remodeling. 6 A major factor contributing to the high incidence of osteoporosis in older women is the reduction in estrogen synthesis that occurs during menopause. Estrogen has wide-ranging effects on the regulation of bone remodeling. Through both direct and indirect effects, it influences calcium bioavailability, the activity of other endocrine factors involved in the regulation of bone remodeling, and the synthesis and activity of various inflammatory cytokines. In rats, surgical menopause (ovariectomy) leads to a selective reduction in the number of vitamin D receptors (VDR) in jejunal but not renal cells. 7 A reduction in the jejunal VDR number results in reduced responsiveness of intestinal cells to vitamin D signaling and, therefore, Affiliations: RC Poulsenand MC Kruger are with the Institute of Food Nutrition and Human Health, Massey University, Palmerston North, New Zealand. Correspondence: MC Kruger, Institute of Food Nutrition and Human Health, Massey University, Private Bag , Palmerston North, New Zealand. m.c.kruger@massey.ac.nz, Phone: , Fax: Key words: daidzein, genistein, isoflavones, osteoblast, osteoclast, osteoporosis. doi: /j x Nutrition Reviews Vol. 66(7):

2 reduced intestinal calcium absorption. As no change occurs in the renal VDR number, vitamin D stimulation of renal calcium excretion is not impaired. This leads to reduced bioavailability of dietary calcium, an essential building block for new bone formation. Estrogen plays a pivotal role in the regulation of the lifecycle and the activity of both osteoblasts and osteoclasts. In humans, some of the effects of estrogen may be direct, as osteoblasts and osteoclasts express both of the known types of estrogen receptor (ER-a and ER-b). 8,9 Estrogen also indirectly regulates osteoblast and osteoclast activity. For instance, estrogen influences the activity of parathyroid hormone (PTH), another endocrine factor involved in the regulation of bone remodeling. PTH has biphasic effects on bone. In low, intermittent doses it promotes osteoblastic bone formation, 10 whereas high, continuous PTH release promotes osteoclastic bone resorption. 11 Estrogen interferes with the ability of PTH to promote bone resorption but does not appear to hinder the anabolic activity of PTH. 12 Estrogen also controls the synthesis of a number of cytokines and growth factors involved in the regulation of bone remodeling. Interaction of estrogen with ER-b results in decreased expression of various inflammatory cytokines, particularly interleukin-1 (IL-1), IL-6, and tumor necrosis factor-a (TNF-a). 13 Pro-inflammatory cytokine expression is increased by free radical reactions normally inhibited by the antioxidant activity of estrogen. 13 Alackof estrogen therefore leads to increased levels of inflammatory cytokines. As a consequence of menopause, the rate of bone resorption increases. Although the bone formation rate also increases, it fails to match that of resorption. The resultant imbalance between bone resorption and formation leads to a reduction in bone mass. PHYTOESTROGENS Several families of molecules are classified as phytoestrogens including lignans, isoflavones, and some flavonoids. The most common dietary source of lignans is linseed (also known as flaxseed), whereas soy is a major source of isoflavones. The focus of this review is on the two most abundant soy isoflavones: genistein and daidzein. Various metabolites of phytoestrogens are also bioactive. Genistein is catabolized by endogenous mammalian enzymes to the bioactive metabolite, 4-ethylphenol, and by enzymes of the intestinal microflora to the non-bioactive metabolite, 4-hydroxyphenyl-2-propionic acid. 14 Unlike genistein, endogenous mammalian enzymes are incapable of metabolizing daidzein. Daidzein is metabolized by the gut microflora to the two bioactive metabolites equol and o-desmethylangolensin (o-dma). 15,16 Equol, o-dma, genistein, and daidzein have both estrogenic and nonestrogenic bioactivity. 17 The activity of 4-ethylphenol has received much less research attention, yet it has been shown to have non-estrogenic bioactivity in reproductive tissue Its possible effect in other tissues is unknown and requires investigation. PHYTOESTROGENS AND BONE A considerable number of studies have been published that have sought to investigate the effect of phytoestrogens on bone metabolism and bone loss in both postmenopausal women and in animal models of postmenopausal bone loss. Epidemiological studies generally suggest a positive association between soy consumption and bone mineral density (BMD). In Asian populations, isoflavone intake hasbeenlinkedwithhigherbmd. 22 One large, epidemiological study found a positive association between total phytoestrogen intake and BMD in the lumbar spine and Ward s triangle (neck of the femur) in postmenopausal but not premenopausal Chinese women. 23 Similarly, in postmenopausal Japanese women, intake of genistein and daidzein has been positively correlated with lumbar spine BMD. 24 A positive association between intake of soy protein and BMD has also been reported in postmenopausal Chinese 25 and Japanese 26 women. Whether this association is due to the presence of isoflavones within the soy products or to soy protein itself is unclear. One study of postmenopausal Korean women found no association between BMD and urinary excretion of genistein, daidzein, or equol (a daidzein metabolite), but it detected a positive association between BMD and urinary excretion of the lignan enterolactone. 27 The lack of association between genistein and daidzein excretion and BMD in this study may reflect the complexity of isoflavone metabolism. Most dietary daidzein and genistein is excreted within 48 h of consumption. 28 Therefore, urinary isoflavone excretion is indicative of acute rather than chronic isoflavone intake. Although changes in urinary isoflavone excretion within an individual may be a useful measure to reflect changes in isoflavone intake within that individual, 29 there is considerable interindividual variation in isoflavone metabolism 30,31 and differences in urinary isoflavone excretion between individuals may not be a reliable predictor of differences in isoflavone intake between individuals. The majority of epidemiological studies performed to date have been conducted in Asian populations; however, there is evidence of racial differences in the ability to metabolize isoflavone. 32 One study reported higher equol and lower o-dma production by Korean women compared to Caucasian women. 32 These differences in isoflavone metabolism may result in different effects of dietary isoflavones on bone. One study, reported lower urinary excretion of NTX (N-telopeptide of type Nutrition Reviews Vol. 66(7):

3 collagen), a marker of bone resorption, in a predominantly Caucasian population of postmenopausal women consuming >1 mg genistein/day. No association with bone mineral density was observed in this study. 33 Although isoflavone consumption of >1 mg/day was relatively high in Caucasian women, in Asian populations, where positive associations between isoflavone intake and BMD have been observed, typical isoflavone intake is >20 mg/day. 23,24 The lack of association with BMD in Caucasian women may be due to the lower habitual isoflavone intake in these women compared to in Asian women. INTERVENTION STUDIES: HUMAN Although a considerable number of intervention studies have been carried out in both humans and animals, these studies have yielded mixed results. This is perhaps not surprising due to the large array of variables known to influence the bioavailability, metabolism and, ultimately, physiological effects of phytoestrogens. In general, isoflavone supplementation studies in premenopausal women tend to report either no effect on bone mass 34 or a possible negative effect in terms of increased circulating concentrations of biochemical markers associated with bone resorption. 35,36 Beneficial effects of isoflavone supplementation on BMD in postmenopausal women have been observed in some but not all studies (Table 1). Both duration and dose of isoflavone supplementation may be important for realizing a significant effect on BMD in postmenopausal women. The composition of isoflavones in the supplement provided may also influence the physiological effect on bone. One well-controlled intervention study by Atkinson et al. 60 reported isoflavone tablets providing 43.5 mg isoflavones per day ameliorated the decrease in lumbar spine BMC and BMD apparent in non-supplemented controls over a 12-month period. Subjects included pre-, peri-, and postmenopausal women in the United Kingdom; however, the vast majority were postmenopausal. Interestingly, the positive effect on BMC and BMD was apparent even though dietary calcium intake was relatively high (approximately 1000 mg/day). 60 The isoflavone supplement used in this study was derived from red clover and contained predominantly formononetin and biochanin. Although formononetin is metabolized to daidzein and biochanin is metabolized to genistein, whether or not the effect on BMD observed in this study was due to the effects of genistein and daidzein is unclear. In a similar study by Morabito et al., 45 significant increases in femoral neck and lumbar spine BMD were observed in early postmenopausal Italian women supplemented with tablets containing 54 mg genistein per day for 12 months. The increase in BMD observed in this study was comparable to that seen in a second study group treated with hormone replacement therapy. In addition, biochemical markers of bone resorption were significantly reduced and markers of bone formation significantly increased in the genistein-supplemented group. 45 However, Chen et al. (2003) observed no effect on BMC or BMD in postmenopausal Chinese women receiving 40 mg per day of daidzein-rich capsules (46.4% daidzein, 38.8% glycitein, and 14.7% genistein). 42 Study duration, time since menopause, and the age of study participants in this study were similar to those in the previously described studies reporting positive effects of isoflavone supplementation on bone mass. The lack of effect on BMD observed in the study of Chen et al. may indicate that some isoflavones are less bone-protective than others, or it may be due to different effects of isoflavones in Asian women compared to Caucasian women. Some studies with shorter durations (8 weeks 6 months) in which isoflavone doses of mg/day have also been used showed no effect of isoflavone supplementation on BMD in postmenopausal women. 44,49,50 This may indicate longer-term supplementation is required in order to elicit observable effects on bone mass in this dose range. In higher doses ( mg per day), isoflavone supplementation has been associated with significant effects on BMD following just 6 months of supplementation. 38,50,57 Several longer-term studies have also shown positive effects on BMD of supplementation with mg isoflavones per day. 42,53 However, other studies of similar duration in which similar isoflavone doses were used, failed to detect any effect of supplementation on BMD. 39,54,56 In a number of the published reports for these studies, the composition of the isoflavone supplement used was not divulged. It is possible that differences in both the types of isoflavones used and the form in which they were provided (glycoside compared to aglycone) influenced the effectiveness of supplementation on bone. One study in humans reported greater systemic bioavailability of glycoside-linked phytoestrogens compared to phytoestrogen aglycones. 61 The glycosylation pattern of the isoflavone also influences the dose, i.e., 20 mg of genistin (the glycosylated form of genistein) is not equivalent to 20 mg of genistein (the aglycone form). Therefore, although it appears that similar doses of phytoestrogens were provided to subjects in the above-listed studies, there may be substantial differences dependent on the form in which the phytoestrogens were provided. Soy protein itself may have beneficial effects on bone metabolism, independent of those of soy isoflavones. 38,39 The presence or absence of soy protein in the supplement provided in intervention studies may influence the observed effect on bone. Decreased bone resorption marker excretion 40,41,45,57 and increased bone formation Nutrition Reviews Vol. 66(7):

4 Table 1 Summary of phytoestrogen intervention studies in humans. Reference Phytoestrogen composition Dose (mg/day) Soy protein Study duration Dietary calcium (mg/day) Outcome Adolescent males Jones et al. (2003) 37 NR 50 NR 6 weeks NR No effect Premenopausal women Geppert et al. (2004) 35 63% genistein, 37% daidzein 52 Yes One menstrual cycle Trial: 1168 Increased ratio of CTX (bone resorption marker): osteocalcin (bone turnover marker) Wangen et al. (2000) 36 55% genistein, 37% daidzein, 8% glycitein Anderson et al. (2002) 34 55% genistein (exact composition not provided) Wangen et al. (2000) 36 55% genistein, 37% daidzein, 8% glycitein 64 Yes ~93 days Base: 848; trial: 1494 Increased DPYD (bone resorption) excretion 90 Yes 12 months Base: 110; trial: 830 No effect 128 Yes ~93 days Base: 848; trial: 592 Increased DPYD (bone resorption marker) excretion at certain stages of menstrual cycle Perimenopausal women Alekel et al. (2000) 38 NR 80.4 Yes 24 weeks Base: 450; trial: >810 Prevented LSBMC and BMD loss Postmenopausal women Gallagher et al. (2004) 39 NR <4 Yes 9 months Base: 641; trial: 1004 Greater BMD Alekel et al. (2000) 38 NR 4.4 Yes 24 weeks Base: 450; trial: >810 No effect Uesugi et al. (2002) 40 50% daidzin, 34% glycitin, 30.9 Yes 4 weeks NR Decreased PYD and DPYD excretion 11% genistin (bone resorption markers) Yamori et al. (2002) 41 ~55% daidzein, 29% glycitein, 15% genistein (mixture of conjugates and aglycones) 37 Yes 10 weeks NR Decreased PYD and DPYD excretion (bone resorption markers) No effect on BMD Chen et al. (2003) 42 NR 40 NR 1 year Trial: 1196 No effect Brooks et al. (2004) 43 61% genistein, 37% daidzein, 41.9 Yes 16 weeks NR No effect 2% glycitein Chiechi et al. (2002) 44 NR 47 Yes 6 months NR No effect Morabito et al. (2002) 45 Genistein 54 No 1 year Base: ; trial: Reduced PYD and DPYD (bone not stated resorption markers) excretion. Increased osteocalcin (bone turnover marker) and BAP (bone formation marker); greater BMD Gallagher et al. (2004) 39 NR 54 or 90 Yes 9 months Base: 641; trial: 1004 No effect Chen et al. (2003) 42 NR 80 NR 1 year Trial: 1127 Increased BMC Albertazzi et al. (2005) 46 Genistein 90 No 6 weeks NR No effect Spence et al. (2005) 47 None 0 Yes 1 month Trial: ~1000 No effect Koyama et al. (2004) 48 NR 22.4 Yes 12 weeks NR Increased BAP (bone formation marker); decreased TRAP (bone resorption marker) 362 Nutrition Reviews Vol. 66(7):

5 Koyama et al. (2004) 48 NR 33.5 Yes 12 weeks NR Increased metacarpal BMD Uesugi et al. (2004) 49 ~49% daidzein, ~38% 40 NR 8 weeks NR Decreased DPYD (bone resorption glycitein, ~13% genistein marker) excretion; no effect on (mixture of conjugates and BMD aglycones) Potter et al. (1998) 50 NR 56 Yes 26 weeks NR No effect 57 Yes 7 weeks Trial: 746 No effect Roughead et al. (2005) 51 54% genistein, 38% daidzein, 8% glycitein Scheiber et al. (2001) 52 NR 60 Yes 12 weeks NR Increased serum osteocalcin (bone turnover marker); decreased NTX excretion (bone resorption marker) Wangen et al. (2000) 36 55% genistein, 37% daidzein, 8% glycitein 65 Yes 93 days Base: 945; trial: 1047 Decreased BAP (bone formation marker) Spence et al. (2005) 47 NR 65 Yes 1 month Trial: 1000 No effect Lydeking-Olsen et al. NR 76 Yes 2 years NR Increased BMC and BMD (2004) 53 Ye et al. (2004) 54 genistein, 41% daidzein, NR 84 NR 24 weeks NR Decreased DPYD excretion (bone resorption marker); no effect on BMD Potter et al. (1998) 50 NR 90 Yes 26 weeks NR Increased BMC and BMD Kreijkamp-Kaspers et al. 53% 99 Yes 12 months Base: 1623; trial: No effect (2004) 55 6% glycitein 1212 Brink et al. (2008) % genistein, 25 35% 110 NR 12 months NR No effect daidzein, 1 5% glycitein Harkness et al. (2004) 57 51% genistein, 40% daidzein, 9% glycitein Nikander et al. (2004) 58 58% glycitein, 36% daidzein, 6% genistein 110 NR 6 months Base: 825; trial: 1200 Decreased type 1 collagen a1 chain helical peptide excretion (bone resorption marker); greater BMD 114 NR 3 months Trial: Reduced PYD and DPYD excretion (bone resorption markers) Dalais et al. (2003) 59 NR 118 Yes 3 months Base: ; No effect trial: NR Ye et al. (2004) 54 NR 126 NR 24 weeks NR Decreased DPYD (bone resorption marker) excretion Wangen et al. (2000) 36 55% genistein, 37% daidzein, 8% glycitein Mixture of pre-, peri-, and postmenopausal women Atkinson et al. (2004) 60 60% biochanin A, 37% formonectin, 2% genistein, 1% daidzein 132 Yes 93 days Base: 945; trial: 1094 Decreased BAP (bone formation marker) 43.5 No 12 months Trial: Reduced BMC and BMD loss at lumbar spine Abbreviations: BAP, bone-specific alkaline phosphatase; BMC, bone mineral content; BMD, bone mineral density; CTX, C-terminal telopeptides of type 1 collagen; DPYD, deoxypyridinoline crosslinks; LSBMC, NR, not reported; NTX, N-terminal telopeptides of type 1 collagen; PYD, pyridinoline crosslinks; TRAP, tartrate-resistant acid phosphatase. Nutrition Reviews Vol. 66(7):

6 marker excretion 60 following isoflavone supplementation have been reported in some studies of postmenopausal women but not in others. 38,43,46,47,51,59 Very few studies have assessed the bioavailability of isoflavones from the supplement provided; however, isoflavone bioavailability may differ considerably among different supplements and in different study populations. For instance, in a recent clinical intervention trial (the PHYTOS study), plasma isoflavone concentrations in French and Italian women were considerably lower than those in Dutch women receiving the same type and quantity of isoflavone-enriched products. 56 The form in which the isoflavones are provided may also influence bioavailability. In the PHYTOS study, in which 110 mg of isoflavones per day were provided in the form of isoflavone-enriched biscuits and cereal bars, plasma isoflavone concentrations in French and Italian women were considerably lower than those observed by Morabito et al. 45 in Italian women receiving 53 mg genistein per day in tablet form or by Lydeking-Olsen et al. 53 in Danish women receiving 76 mg isoflavones per day from soy milk. No effect of isoflavone supplementation on BMD was observed in the PHYTOS study 56 ; however, positive effects on BMD were observed in the Morabito et al. 45 and Lydeking-Olsen et al. 53 studies. Although considerable inter-individual variability in plasma isoflavone concentrations was observed in all three studies, it is possible that the food matrix in which the isoflavone supplement is delivered influences isoflavone bioavailability. The lower plasma isoflavone concentrations observed in the PHYTOS study may have contributed to the lack of effect of isoflavone supplementation on BMD in this study. Further investigation into the relative bioavailability of isoflavones from tablets, foods naturally rich in isoflavones, and isoflavone-enriched products is required as differences in bioavailability may considerably impact the physiological effect of supplementation on bone. Differences between studies in terms of the true dose of phytoestrogens provided as well as in the bioavailability of phytoestrogens from the supplement used may account for at least some of the discrepancies in the reported effects of isoflavone supplementation on bone in humans. There is considerable inter-individual variation in isoflavone metabolizing ability. In Western societies only approximately 33% of the population is capable of producing equol 31 and 80 90% capable of producing o-dma. 62 The composition of the gut microflora is a major factor governing daidzein metabolism. 63 There is some indication that individuals who consume high amounts of isoflavones develop higher amounts of flavonoid-metabolizing bacteria in the colon 64 and, therefore, may produce relatively more equol and/or o-dma than those with a lower isoflavone intake. Age, race, 62 and dietary components such as the carbohydrate content of the diet, 65 presence of probiotics, 66 total amount of fat in the diet, 67 and type of dietary fat 68,69 also influence phytoestrogen metabolism. As equol has greater estrogenic and antioxidant activity than daidzein 15 and 4-ethylphenol is more effective than genistein in modulating prostaglandin synthesis, 21 the extent of metabolite formation from genistein and daidzein is likely to have a major role in determining the physiological effects of isoflavone consumption. Differences among study populations in terms of isoflavone-metabolizing ability may also impact trial outcome considerably. In some cases, poor study design has hindered the interpretation of study results. In several studies, the confounding effects of other variables on bone mass, notably dietary calcium intake, have been inadequately controlled or accounted for. In a number of cases, calcium intake in the study population has not been reported. 40,41,43,44,53,54 In other studies, calcium intake was substantially altered during the trial period, 38,55,57 possibly masking any physiological or metabolic change in bone caused by the phytoestrogens. INTERVENTION STUDIES: ANIMAL There are a number of similarities between ovariectomyinduced bone loss in rats and postmenopausal bone loss in humans. 70,71 As a consequence, the ovariectomized rat is widely used as a model for postmenopausal bone loss.a number of studies have examined the effects of isoflavones on bone in intact and ovariectomized rats as well as in other ovariectomized animal models. These studies are summarized in Table 2. Interpretation of the results from intervention studies in animals is hindered by the range of different ways in which the dose of isoflavones used has been reported in the literature. In some cases, the dose of isoflavones is reported as the amount of isoflavones per kilogram of diet; in others, as the amount of isoflavones consumed per kilogram of rat body weight. Although both are valid methods of reporting dose, it is difficult to make comparisons between trials using the two different conventions unless sufficient supporting information is provided, such as the amount of food consumed by the animals per day or the body weight of the animals. In order to aid in comparing the results of different trials, this review, where possible, discusses isoflavone intakes in terms of the amount consumed per kilogram of animal body weight. Intact growing rats Studies in intact growing rats have yielded mixed results. One study in intact weanling rats showed no effect on 364 Nutrition Reviews Vol. 66(7):

7 BMD of 60 days of supplementation with soy isoflavones (0.046% of diet, composition, or actual amount of isoflavones consumed not reported). 86 However, a slight reduction in bone mineral content but not density was observed in another study of female rats treated for 2 years with genistein (0.05% of diet). 87 Although the percentage of isoflavones in the diet was similar in these two studies, the actual amount of diet consumed by rats per day was not reported in either study. It is therefore not possible to determine if animals in the two studies were receiving the same dose of isoflavones per day. Beneficial effects of isoflavone treatment on bone mass in growing animals have also been reported. Increased tibial BMD following 21 days of subcutaneous injection with genistein (5 mg genistein/kg body weight/ day) was observed in one study in 2-month-old, nonovariectomized rats. 74 Dietary supplementation for 3 months with 18 mg soy isoflavone aglycones/kg body weight/day (containing % genistein and % daidzein) was associated with slight increases in BMD in the lumbar spine in 3-month old, nonovariectomized rats. 88 In the same study, there was no significant effect of supplementation with 10 mg aglycones/kg body weight/day on BMD. 88 As genistein is metabolized by endogenous enzymes in the stomach and intestine to compounds with either no known bioactivity or non-estrogenic bioactivity, subcutaneous injection is likely to result in more intact genistein in circulation compared to oral administration. If genistein itself rather than one of its metabolites is bioactive in bone, a lower dose of genistein may be required to elicit boneprotective effects when genistein is administered subcutaneously as opposed to orally. Although the genistein metabolite 4-ethylphenol is bioactive, whether it has bioactivity in bone is unknown and requires further investigation. One study in ovariectomized rats found that daidzein had greater bone-protective effects than genistein when administered as an oral supplement. 90 Whether the same is true in intact rats is unknown but, if so, may at least partially explain the lack of consensus in results obtained from intervention studies in growing rats. Ovariectomized rats Unlike in humans, long bones in rats continue to grow well past the attainment of maturity. During growth, two processes operate in bone: bone modeling, which results in bone growth, and bone remodeling, which leads to maintenance and repair of bone. In female rats, longbone growth ceases at approximately 6 months of age. 91,92 The post-ovariectomy effect of isoflavones on bone may therefore differ in growing rats (<6 months of age) compared to skeletally mature rats ( 6 months of age). Growing ovariectomized rats. The dose required to have a beneficial effect on BMD in ovariectomized animals may be higher than that required in non-ovariectomized animals, as subcutaneous injection with 5 mg genistein/kg body weight for 21 days had no effect on tibial BMD in 2-month-old ovariectomized rats but, as previously discussed, was associated with increased tibial BMD in 2-month-old intact rats. 74 There is some indication that isoflavones may have a biphasic effect on bone. In ovariectomized mice, subcutaneous injection with 0.4 mg isoflavones/day increased femoral BMD; however, a dose of 0.7 mg/day had no effect on BMD. 78 Increased femur calcium content has been observed following 6 weeks of supplementation with approximately 4 mg of isoflavones/kg body weight (80 ppm in diet) in growing ovariectomized rats fed a low-calcium diet (0.1% calcium in diet). 76 Higher isoflavone intakes may be required in order to achieve a significant effect on bone in animals fed a calcium-adequate diet, as no effect on bone was observed in trials of similar duration in which animals were supplemented with isoflavones in doses ranging from 3 to 7 mg/kg rat body weight/day. 73,75 However, isoflavone supplementation at the level of mg/kg rat body weight/day has been shown to increase bone mineral content or density in growing, ovariectomized rats fed a calcium-adequate diet, 73,77 but not when supplementation commences several weeks following ovariectomy. 72 Skeletally mature ovariectomized rats. In 6-month-old ovariectomized rats consuming a low-calcium diet (0.24% calcium in diet), greater femur BMD and strength were observed following 13 weeks of supplementation with 20, 40, or 80 mg of isoflavones per kilogram rat body weight per day. 83 However, no such beneficial effect on BMD was observed in a similar study of 6-month-old rats when supplementation commenced 12 weeks following ovariectomy. 82 This may indicate that isoflavones prevent, but cannot reverse, ovariectomy-induced bone loss. Whether isoflavones are effective in minimizing ovariectomy-induced bone loss in skeletally mature animals fed a calcium-adequate diet remains to be determined. One study found no effect on BMD of 8 weeks of isoflavone supplementation, with or without soy protein, in 6-month-old ovariectomized rats fed a calciumadequate diet (0.54% calcium in diet). 81 However, neither the daily isoflavone intake nor isoflavone intake per kilogram of rat body weight could be determined from data provided in the published report. It is therefore unclear whether the dose of isoflavones used in this latter study was comparable to those used in studies reporting favorable effects on BMD. As is the case with human intervention studies, in some reports of intervention studies conducted in animals, the composition of the isoflavone supplement Nutrition Reviews Vol. 66(7):

8 Table 2 Summary of phytoestrogen intervention studies in animals. Reference Phytoestrogen composition Soy protein? Growing (2 3-month-old) OVX rat Arjmandi et al. (1998a) 72 70% genistin, 1% genistein, 28% daidzin, 0.5% daidzein Arjmandi et al. (1998a) 72 70% genistin, 1% genistein, 28% daidzin, 0.5% daidzein Arjmandi et al. (1998b) 73 70% genistin, 1% genistein, 28% daidzin, 0.5% daidzein Dose (per day) Mode Study length % Ca in diet Yes ~0.84 mg Diet 65 days (35 days post-ovx) Yes ~8.39 mg Diet 65 days (35 days post-ovx) Outcome NR No effect NR No effect Yes ~0.84 mg Diet 35 days 0.4 No effect 3 4 mg/kg body weight Fanti et al. (1998) 74 Genistein No 1 mg/kg body weight SC 21 days 0.6 No effect Deyhim et al. (2003) 75 NR NR ~0.96 mg Diet 40 days 0.5 No effect 3.5 mg/kg body weight Kim et al. (2005) 76 50% genistein, 38% daidzein, 12% glycitein No 80 ppm in diet. ~4 mg/kg body weight Diet 6 weeks 0.1 Greater bone calcium content Fanti et al. (1998) 74 Genistein No 5 mg/kg body weight SC 21 days 0.6 Increased serum osteocalcin (bone turnover marker) Deyhim et al. (2003) 75 NR NR ~1.92 mg Diet 40 days 0.5 No effect 7 mg/kg body weight Kim et al. (2005) 76 50% genistein, 38% daidzein, 12% glycitein Arjmandi et al. (1998b) 73 70% genistin, 1% genistein, 28% daidzin, 0.5% daidzein Lee et al. (2004) % daidzin, 34.2% glycitin, 11.5% genistin, 1.7% glycitein, 0.6% daidzein, 0.4% genistein No 160 ppm in diet. ~8 mg/kg body weight Yes ~8.4 mg Diet 35 days mg/kg body weight (5 weeks) Diet 6 weeks 0.1 Greater lumbar spine dry weight 0.4 Greater BMD No 6.25 g/kg diet ~50 mg/kg body weight Diet 16 weeks 0.4 Greater BMD Growing (2-month-old) OVX mice Ishimi et al. (2000) 78 Genistein No 0.4 mg SC 4 weeks 0.5 Greater BMD Ishimi et al. (2000) 78 Genistein No 0.7 mg SC 4 weeks 0.5 Greater BMD Ward et al. (2005) 79 Daidzein No 200 mg/kg diet. ~22 mg/kg body weight Wu et al. (2004) % daidzin, 13% glycitin + glycitein, 4.6% genistin Diet 3 weeks NR No effect NR 160 mg/kg body weight Diet 6 weeks NR Greater BMD Mature (6 7-month-old) OVX rat Cai et al. (2005) 81 NR Yes 0.4 mg isoflavones/g diet Diet 8 weeks 0.54 No effect Cai et al. (2005) 81 NR Yes 0.2 mg isoflavones/g diet Diet 8 weeks 0.54 No effect Cai et al. (2005) 81 NR Yes Negligible Diet 8 weeks 0.54 No effect Cai et al. (2005) 81 NR No 0.3 mg isoflavones/g diet Diet 8 weeks 0.54 No effect Cai et al. (2005) 81 NR No 0.8 mg isoflavones/g diet Diet 8 weeks 0.54 No effect Picherit et al. (2001a) % genistin, 44.8% daidzin, 9.5% glycitin Picherit et al. (2001a) % genistin, 44.8% daidzin, 9.5% glycitin No 20 mg/kg body weight Diet 84 days (80 days post-ovx) No 40 mg/kg body weight Diet 84 days (80 days post-ovx) 0.24 No effect 0.24 Lower plasma osteocalcin (bone turnover marker) and urinary DPyd excretion (bone resorption marker) 366 Nutrition Reviews Vol. 66(7):

9 Picherit et al. (2001a) % genistin, 44.8% daidzin, 9.5% glycitin Picherit et al. (2001b) % genistin, 44.8% daidzin, 9.5% glycitin Picherit et al. (2001b) % genistin, 44.8% daidzin, 9.5% glycitin Picherit et al. (2001b) % genistin, 44.8% daidzin, 9.5% glycitin Aged (11 12-month-old) OVX rat Blum et al. (2003) :1 ratio of genistein and daidzein in diet No 80 mg/kg body weight Diet 84 days (80 days post-ovx) 0.24 No effect No 20 mg/kg body weight Diet 91 days 0.24 Decreased plasma osteocalcin (bone turnover marker) No 40 mg/kg body weight Diet 91 days 0.24 Decreased plasma osteocalcin (bone turnover marker); greater BMD No 80 mg/kg body weight Diet 91 days 0.24 Decreased plasma osteocalcin (bone turnover marker); greater BMD Yes Minimum 451 mg isoflavones/kg diet Diet 3 months NR Greater BMD Picherit et al. (2000) 85 Genistein No 10 mg/kg body weight Diet 90 days 0.23 Greater BMD Picherit et al. (2000) 85 Daidzein No 10 mg/kg body weight Diet 90 days 0.23 Greater BMD Growing sham-operated rat Fanti et al. (1998) 74 Genistein No 5 mg/kg body weight SC 21 days 0.6 Increased serum osteocalcin (bone turnover marker); greater BMD Growing intact rat James et al. (2002) 86 NR Yes 0.046% of diet Diet 60 days 0.5 No effect James et al. (2002) 86 NR No 0.046% of diet Diet 60 days 0.5 No effect Hotchkiss et al. (2005) 87 Genistein No 5 ppm Diet 2 years 1.15 Lower BMC and BA Hotchkiss et al. (2005) 87 Genistein No 100 ppm Diet 2 years 1.15 Increased urinary PYD excretion (bone resorption marker). Lower BMC and BA Hotchkiss et al. (2005) 87 Genistein No 500 ppm Diet 2 years 1.15 Increased urinary PYD excretion (bone resorption marker). Lower BMC and BA Nakai et al. (2005) 88 52% genistein, 39% daidzein, 9% glycitein Nakai et al. (2005) 88 52% genistein, 39% daidzein, 9% glycitein Nakai et al. (2005) 88 62% genistein, 34% daidzein, 4% glycitein Nakai et al. (2005) 88 62% genistein, 34% daidzein, 4% glycitein Aged sham-operated rat Blum et al. (2003) :1 ratio of genistein and daidzein in diet OVX monkey Register et al. (2003) 89 71% genistein, 24% daidzein, 5% glycitein No 8.9 mg/kg body weight Diet 14 weeks 0.85 No effect No 18.1 mg/kg body weight Diet 14 weeks 0.85 Slightly greater BMD Yes 10.1 mg/kg body weight Diet 14 weeks 0.85 Greater BMD Yes 20.2 mg/kg body weight Diet 14 weeks 0.85 Decreased urinary DPYD (bone resorption marker); greater BMD Yes Minimum 451 mg isoflavones/kg diet Diet 3 months NR No effect Yes mg isoflavones/monkey; ~10 11 mg/kg body weight Diet 3 years 830 mg per day Decreased serum CTX (bone resorption marker) and ALP (bone formation marker) at 3 months Abbreviations: ALP, alkaline phosphatase; BA, bone area; BMC, bone mineral content; BMD, bone mineral density; CTX, C-terminal telopeptides of type 1 collagen; DPYD, deoxypyridinoline crosslinks; NR, not reported; OVX, ovariectomized; PYD, pyridinoline crosslinks; Sham, sham-operated; SC, subcutaneous injection. Nutrition Reviews Vol. 66(7):

10 used was not provided. This is despite the finding reported in 2000 by Picherit et al. 90 that daidzein in a dose of 10 mg/kg rat body weight/day was more effective than genistein at maintaining BMD in the lumbar spine and femur in 12-month-old ovariectomized rats consuming a low-calcium diet (0.23% calcium in diet). The daidzein content of an isoflavone supplement may influence the effect of supplementation on bone. MECHANISMS OF ACTION Phytoestrogens are best known for their ability to mimic the activity of mammalian estrogens; however, they can act as either estrogen agonists or antagonists depending on biological conditions. 93 In addition, they have a number of other biological effects, independent of those of estrogen. Estrogenic activity of phytoestrogens Phytoestrogens bind to both known subtypes of the ER (ER-a and ER-b); however, they have a much greater affinity for ER-b than ER-a. 94 The binding affinities of genistein and daidzein relative to 17b-estradiol for ER-a are 0.7% and 0.2%, respectively. In comparison, the binding affinities of genistein and daidzein for ER-b are approximately 13% and 1%, respectively, of that of 17bestradiol. 95 Phytoestrogens interact with the estrogen receptors in a different manner than endogenous estrogens. Whereas 17b-estradiol has a lipophylic region, which is thought to influence receptor-binding, genistein and daidzein lack this region. Interaction of genistein rather than mammalian estrogens with ER-b leads to changes at a different position of the transactivation helix resulting in lower estrogenic activity. 94 Phytoestrogens also stimulate transcription of ER-a and ER-b. 65 Daidzein has been shown to selectively enhance nuclear ER-b levels. This is in contrast to 17b-estradiol, which enhances expression of both ER-a and ER-b. 96 Anti-estrogenic effects Phytoestrogens also act in a number of ways to oppose the action of mammalian estrogens and other sex hormones. They stimulate the synthesis of sex hormone binding globulin resulting in an increase in the amount of proteinbound, and therefore unavailable, estrone and estradiol in the blood. 65 Phytoestrogens also inhibit several enzymes involved in the metabolism of sex hormones. These include: 5a-reductase (converts testosterone to dihydrotestosterone), 17b-hydroxysteroid dehydrogenase (regulates interconversion of testosterone and androstenedione as well as 17b-estradiol and estrone) and the human P450 aromatase system (involved in estrone metabolism). 65 Genistein, daidzein, and equol compete with estradiol for nuclear type II estrogen-binding sites and, as a result, regulate estrogen-stimulated growth and proliferation. 97 Other effects Both genistein and daidzein are weak antioxidants and inhibit angiogenesis. 65 Genistein is a tyrosine kinase inhibitor and this may be one of the mechanisms by which it impedes cancer cell growth. 65 Other enzymes known to be inhibited by genistein include topoisomerases I and II and protein histidine kinase. 97 Genistein also inhibits leptin secretion by adipocytes, which may impact bone metabolism. 98 Effect on calcium balance In vitro, genistein and daidzein reduced transepithelial calcium absorption in the human intestinal-like Caco-2 cell line when cells were grown in the presence of estrogen but had no effect on calcium absorption in the absence of estrogen. 99 In 3-month-old rats, supplementation with soy protein containing isoflavones (but not soy protein alone) prevented the ovariectomy-induced reduction in calcium transport in duodenal and colonic cells in in vitro transport experiments. However, no effect of soy protein supplementation, with or without isoflavones, on urinary calcium excretion or circulating calcium levels was observed. 100 Similarly, in postmenopausal Caucasian women, no effect of soy protein, with or without isoflavones, was evident on calcium absorption, excretion, or balance. 47 The overall effect of soy isoflavones on intestinal calcium absorption in vivo therefore appearstobeminimal. Effect on osteoblastogenesis and osteoblast activity Both genistein and daidzein stimulate osteoblast proliferation, differentiation, and activation by an ER-dependent mechanism. 96,101,102 Core binding factor-1 (Cbfa-1) and bone morphogenic protein-2 (BMP-2) are transcription factors involved in the differentiation of osteoblasts from progenitor cells BMP-2 106,107 and Cbfa-1 96,102 synthesis is upregulated by daidzein and genistein. Both isoflavones also promote bone nodule formation in vitro. Whilst the effects of genistein on bone nodule formation appear to be ER-dependent, daidzein may act in an ER-independent manner. 108 Genistein 109 and daidzein 110 also activate peroxisome proliferator activator receptors (PPARs). PPAR activation can modulate ER activity and the balance between PPAR and ER activation may govern the balance between adipogenesis and osteoblastogenesis Nutrition Reviews Vol. 66(7):

11 Daidzein dose-dependently activates PPAR-a, PPAR-d, and PPAR-g. 110 Daidzein has a biphasic effect on osteogenesis. At low concentrations it stimulates osteoblast differentiation but at high concentrations it promotes adipogenesis. The mechanism may involve PPAR activation. Daidzein-induced activation of PPAR-g increases along with increasing daidzein dose. The effects of daidzein on PPAR-d are biphasic and PPAR-d activation is minimal at high daidzein concentrations. PPAR-d activation is believed to be a major contributor to the mechanism by which daidzein promotes osteogenesis. In one in vitro study, daidzein-mediated activation of PPAR-a had no effect on osteogenesis or adipogenesis, but it inhibited daidzein-induced ER-mediated transcriptional activity. Daidzein-mediated activation of PPAR-d stimulated osteogenesis and upregulated ER-mediated transcriptional activity but had no effect on adipogenesis, whereas activation of PPAR-g by daidzein inhibited osteogenesis and ER-mediated daidzein activity and stimulated adipogenesis. 110 Genistein also acts as a PPAR-g ligand and high concentrations of genistein result in PPAR-g-stimulated adipogenesis at the expense of osteogenesis. 109 Conversely, low concentrations of genistein promote osteogenesis 109 and in bone marrow stromal cells, low concentrations of genistein have been shown to decrease PPAR-g protein expression during adipogenesis, thereby inhibiting adipocyte formation. The mechanism appears to be ER-mediated and involve upregulation of transforming growth factor b1 protein levels. 111 Genistein has also been shown to inhibit PPAR-g activity in human osteoblasts through activation of ER-a. 112 The effect of genistein on PPAR-d is unknown. Although genistein also activates PPAR-a, 113 no direct effect of genistein-mediated PPAR-a activation on bone cells has been documented. Soybean isoflavones, in conjunction with saponins (a family of plant-derived triterpenes and steroids conjugated with either alkaloids or glycosides), increased serum g-carboxylated osteocalcin concentrations in healthy men and women. 114 g-carboxylation of osteocalcin is usually performed by vitamin K and is essential for enabling hydroxyapatite binding in the bone matrix. 114 However, whether the increase in serum g-carboxylated osteocalcin concentration was due to the effects of isoflavones or of saponins is unknown. Effect on osteoclastogenesis and osteoclast activity Low-dose genistein (10-8 M) decreased osteoclast numbers in bone marrow culture by decreasing osteoclast viability. Higher concentrations of genistein (10-5 M) attenuated osteoclast formation. 115 In RAW264.7 cells, a cell line capable of differentiating into osteoclasts, genistein, daidzein, and 17b-estradiol stimulate ER-a expression and promote proliferation but inhibit multinucleation (and therefore differentiation into the mature osteoclast phenotype). 116 One of the major pathways governing osteoclastogenesis involves a triad of proteins including a receptor (receptor activator of nuclear factor kappa B [RANK]), a ligand (receptor activator of nuclear factor kappa B ligand [RANKL]), and a decoy receptor (osteoprotegerin [OPG]). 117,118 RANKL and OPG are expressed by osteoblasts 119 (as well as some other cell types such as T-cells 120 ), whereas RANK is expressed by osteoclasts. 121 RANK is a membrane-bound receptor, whereas OPG is soluble. RANKL exists in both membrane-bound and soluble forms. Binding of RANKL to RANK stimulates osteoclastogenesis. 122 However, binding of RANKL to OPG prevents RANKL-RANK binding and therefore indirectly inhibits osteoclastogenesis. 123 The relative levels of RANK, RANKL, and OPG are important for controlling osteoclastogenesis. In osteoblasts isolated from trabecular bone from young piglets, daidzein increased secretion of both OPG and soluble RANKL (srankl) and increased concentration of membrane-bound RANKL by an ER-mediated mechanism. 96 This indicates daidzein regulates expression of both RANKL and OPG; however, the overall effect of these daidzein-induced changes on RANKL-induced osteoclastogenesis is unclear. Daidzein has been shown to promote apoptosis of osteoclast progenitors by an ER-mediated mechanism. 124 Genistein modulates expression of both RANKL and OPG. The mechanism appears to be linked to genistein s ability to inhibit topoisomerase-ii activity. 125 In postmenopausal women supplemented with genistein for 12 months, the ratio of srankl:opg in serum was significantly lower than in non-supplemented controls. 126 This may indicate that genistein inhibits RANKL-induced osteoclastogenesis in postmenopausal women. Proinflammatory cytokines stimulate osteoclastogenesis by both RANKL-dependent 127 and RANKLindependent mechanisms. 128 Genistein and daidzein may inhibit pro-inflammatory cytokine synthesis as serum concentrations of the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a) were found to be significantly lower in postmenopausal women consuming a soy-supplemented diet than in women not receiving the soy isoflavone supplement. 129 Similarly, a significant reduction in serum IL-1b and TNF-a concentrations were observed in ovariectomized rats treated with genistein. 130 In vitro, genistein and daidzein inhibit synthesis of the pro-inflammatory cytokine IL-6 by MC3T3-E1/4 osteoblast-like cells They have also been shown to inhibit the rise in production of the pro-inflammatory eicosanoid prostaglandin E2 (PGE2) following exposure to TNF-a in MC3T3-E1/4 cells. 133 Although both IL-6 and PGE2 are produced by Nutrition Reviews Vol. 66(7):

12 cells other than osteoblasts, inhibition of their synthesis in osteoblasts may indicate that genistein and daidzein also inhibit synthesis of pro-inflammatory signaling molecules in other cell types. As well as influencing osteoclastogenesis, genistein and daidzein appear to influence osteoclast activity. Both genistein and daidzein have been found to inhibit inward rectifier K+ channels in osteoclasts, leading to membrane depolarization, intracellular influx of Ca2+ and inhibition of osteoclast-mediated bone resorption. 134 Very few studies have examined the effect of the bioactive metabolites of genistein and daidzein on osteoblasts or osteoclasts. In high concentrations (>10-7 M), equol has been shown to inhibit differentiation of RAW cells into osteoclasts. 135 TheeffectsofDMAand 4-ethylphenol on bone cells have yet to be examined. CONCLUSION In vitro, genistein and daidzein have both estrogenic and non-estrogenic bioactivity, which can influence osteoblastogenesis and osteoclastogenesis, as well as the activities of both osteoblasts and osteoclasts. There are a number of differences in the bioactivities of genistein and daidzein at the cellular level, and these may translate into different physiological effects in vivo. The effects of the bioactive metabolites of genistein and daidzein (4-ethylphenol, equol, and o-dma) on osteoblasts and osteoclasts are largely unknown and this is an area requiring further investigation. In particular, it is currently unknown whether 4-ethylphenol has any bioactivityinbone. Differences in the design of intervention studies in both animals and humans have hindered the interpretation of results obtained from studies conducted thus far. Soy isoflavones may influence bone metabolism and bone density in vivo under some circumstances in both animals and humans. However, more work is required in order to determine the extent of this effect, the circumstances under which isoflavone supplementation is efficacious, and the dose and composition of the isoflavone supplement required. There are a number of key lessons from studies conducted thus far that should be taken into account in the design of future research in order to aid in the interpretation of results. 1) The composition of the isoflavone supplement used in a trial needs to be clearly established. 2) The form in which the phytoestrogen is provided (i.e., aglycone or glycoside) needs to be stated and the dose of glycosylated isoflavones administered in an intervention trial needs to be stipulated in terms of aglycone equivalents. 3) Serum or plasma concentrations of the phytoestrogens and their bioactive metabolites should be measured to obtain an estimate of the bioavailability of the isoflavone supplement and the degree of formation of bioactive metabolites within the specific study population. REFERENCES 1. Fraser WD. The burden of osteoporosis and the case for disease management. Dis Manag Health Out. 2004;12: Consensus Development Conference. Prophylaxis and treatment of osteoporosis. 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Identification of isoflavone metabolites dihydrodaidzein, dihydrogenistein, 6 -OH-O-dma, and cis-4-oh-equol in human urine by gas chromatography-mass spectroscopy using authentic reference compounds. Anal Biochem. 1999;274: Mueller SO, Simon S, Chae K, Metzler M, Korach KS. Phytoestrogens and their human metabolites show distinct agonistic and antagonistic properties on estrogen receptor {alpha} (ER{alpha}) and ER{beta} in human cells. Toxicol Sci. 2004;80: Woclawek-Potocka I, Acosta TJ, Korzekwa A, et al. Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms. Exp Biol Med. 2005;230: Nutrition Reviews Vol. 66(7):